Atherosclerosis is the progressive narrowing of the lumen (inner passageway) of arterial blood vessels by layers of plaque (fatty and fibrous tissues). Atherosclerosis can occur in any artery. In coronary arteries it may result in heart attacks; in cerebral arteries it may result in strokes; and in peripheral arteries it may result in gangrene of the legs and feet.
Atherosclerosis is the single largest medical problem currently facing the United States and other developed countries. Approximately 40 million people in the United States are at risk for atherosclerosis. However, only 6 million people in the United States show overt signs of the disease. The rest remain undiagnosed until the disease manifests itself symptomatically, in the worst case as heart attack or stroke. Heart attack and stroke, respectively, represent the first and third leading causes of death in United States. Over 500,000 people die of heart attacks every year and a significant sub-group of these patients expire without warning.
The endothelium is located between the blood and arterial tissue and serves as a barrier against the accumulation of blood components in the vascular wall. Formation of atherosclerotic lesions in the sub-endothelium is associated with major coronary artery disease and stroke. The causes and detection of such lesions have been intensely investigated.
Endothelial injury is believed to be an initial step in the formation of the atherosclerotic lesions and may be caused by hemodynamic strain, hypercholesterolemia, hypertension and immune complex disease. Endothelial injury leads to cholesterol accumulation and intimal thickening, cellular proliferation, and formation of connective tissue fibers. IgG and complement factor C3 accumulation in injured endothelial cells and nonendothelialized intima has been observed. Mononuclear phagocytes derived from blood are also part of the cell population in atherosclerotic lesions. The mechanism of plaque formation is not fully known. However, a probable mechanism is that fatty deposits lead to an influx of macrophages, which in turn are followed by T cells, B cells, and antibody production.
A variety of soluble proteins have been extracted from human atherosclerotic plaque, including IgA, IgG, IgM, B1C(C3), alpha.sub.1 -antitrypsin, alpha.sub.2 -macroglobulin, fibrinogen, albumin, LDL, HDL, alpha.sub.1 -acid glycoprotein, .beta..sub.2 -glycoprotein, transferrin and ceruloplasmin. The diseased intima was also found to contain a small amount of tissue-bound IgG, IgA and B1C [Hollander, W. et al., Atherosclerosis, 34:391-405 (1979)]. IgG has been determined in lesions and adjacent endothelial tissue [Parums, D. et al., Atherosclerosis, 38:211-216 (1981), Hansson, G. et al., Experimental and Molecular Pathology, 34:264-280 (1981), Hannson, G. et al., Acta Path. Microbiol. Immunol. Scand. Sect. A., 92:429-435 (1984)]. However, the origin, function and binding properties of the immunoglobulins in the atherosclerotic and associated tissue are not well characterized. Anti-low density lipoprotein (LDL) autoantibodies are reported to be higher in patients of vascular disease, suggesting that they are associated in some way with atherosclerotic manifestations. However, no causal relationship between these autoantibodies and atherosclerotic plaque has been established. [Szondy, E. et al., Mechanisms of Aging and Development, 29:117-123 (1985)].
A wide variety of immunoassays have been developed for determining the presence and amount of antigenic and non-antigenic materials in diverse body fluids and tissues. Total immunoglobulin and IgE immunoassays are described in U.S. Pat. Nos. 3,720,760 and 4,444,879. IgG allotype immunoassays are described in Russian Patent 649,433. ELISA immunoassays are described by Maggio, et al. [Enzyme-Immunoassay, Boca Raton: CRC Press pp 172-176 (1980)]. However, prior to this invention, no immunoassay suitable for determining the presence of atherosclerotic plaque has been known.
Although atherosclerosis is generally a diffuse disease, human coronary atherosclerosis lends itself to bypass procedures because the major site of plaque formation is usually proximally distributed. As a result, direct coronary artery bypass has become the most frequently selected form of myocardial revascularization. The aorta-coronary artery vein graft or the internal mammary artery graft have become technically standardized and have high long-term potency rates. These long-term results, however, can be compromised by progressive atherosclerosis distal to the graft anastomosis. Other cases are inoperable because of distal disease. Previously, distal lesions have been ignored or, in selected cases, treated by endarterectomy although neither approach has proved entirely satisfactory.
Most existing procedures for the diagnosis and treatment of atherosclerosis are invasive, costly, and of limited effectiveness in a significant percentage of patient cases.
Prior to the subject invention, radioimaging of atheroscelerotic plaque using an antibody which specifically binds to an atherosclerotic plaque specific antigen was unknown, although radioimaging of aged venous thrombi with fibrin-specific monoclonal antibodies labeled with a radioactive moiety has been reported [Rosebrough, S. et al., Radiology 162:575-577 (February, 1987)].
Radioimaging thrombi with radiolabeled monoclonal antibodies to platelets was first described by Peters, A., et al. [British Medical Journal, 293:1525-1527 (December, 1986)]. DTPA-coupled antibodies radiolabeled with metallic radionuclides has been described by Hnatowich, D., et al. [Journal of Immunological Methods, 65:147-157 (1983)].
NMRI, ultrasound and X-ray imaging with metal chelates are described in U.S. Pat. No. 4,647,447. In addition, antibody coupling of metal chelates is mentioned at column 7, line 42. Monoclonal antibodies labeled with polymeric paramagnetic chelates and their use in NMRI methods have also been described [Shreve, P. et al., Magnetic Resonance in Medicine, 3:336-340 (1986) and Brady, T. et al. in Proceedings of the Society of Magnetic Resonance in Medicine, Second Annual Meeting, Soc. of Magnetic Resonance in Medicine, Inc., San Francisco, p. 10, (1983), referenced by Koutcher, J. et al., J. Nucl. Med., 25:506-513 (1984)].
U.S. Pat. No. 4,343,734 (Lian et al.) describes gamma-carboxyglutamic acid (GLA) specific antibodies which can be labeled with fluorescein for immunofluorescence staining of tissue to determine the presence therein of GLA. GLA specific antibodies bind with GLA present in advanced atherosclerotic plaque having calcium deposits. Lian et al. report that GLA is not found in uncalcified plaques and that GLA is found in cardiac valves and aortas, and in circulating proteins such as prothombin, clotting factors VII, IX and X, Protein C and Protein S. However, the GLA binding antibodies of Lian et al. do not selectively bind to atherosclerotic plaque.
The atherosclerotic plaque antibodies of the subject invention bind to all stages of atherosclerotic plaque including non-calcified stages, and do not selectively bind with GLA.
The concept of plaque enhancement by application of a stain has been reported [Spears, J. et al., J. Clin. Invest, 71:395-399 (1983)]. These stains mark the plaque surfaces with a fluorescent compound. Plaque destruction by photoactivation of hematoporphyrin derivatives using an intraluminal. laser-transmitting optical fiber has been suggested [Abela, G. et al., Am. J. Cardio., 50:1199-1205 (1982)]. Moreover, tetracycline stains have also been suggested. [Murphy-Chutorian, D. et al., Am. J. Cardiol., 55:1293-1297 (1985)].
The above-identified stains were selected for their ability to bind to components of the atherosclerotic plaque. In principal, the stain absorbs laser light concentrating the light at the stained surface. Some staining of healthy tissue occurs causing stain associated damage to the surrounding tissue. Because laser wavelength is limited to the absorption wavelength of the stain, chromophores offering optimum absorption of laser must be used to provide most controlled ablation.
In recent years, lasers have been used increasingly in microsurgery, both as scalpels and as coagulating instruments. Because of their ability to produce relatively bloodless incisions of great precision, as well as focal coagulation, they have been particularly useful in microsurgical procedures in the eye, central nervous system, nasal passages, cervix, gastrointestinal tract, skin, muscle, and even in small vessels.
In vivo experiments with heart and arterial tissue from human cadavers have demonstrated the feasibility of vaporizing or etching away plaque on diseased surfaces. UV-wavelengths were found to offer more precision. Laser treatment of plaque in live animals was less precise, causing damage and perforation of surrounding healthy tissue. [Gerrity, R. et al. J. Thorac. Cardiovasc. Surg., 85:409-421 (1983); Lee, G. et al., Am Heart J., 105:885-889 (1983); Lee, G. et al., Am. Heart J., pp 777-778. (August 1984); Lee, G. et al., Am. Heart J., 108:1577-1579 (1984); Lee, G. et al., Am. J. Cardiology, 53:290-293 (1984); Linsker, R. et al., Lasers in Surgery and Medicine, 4:201-206 (1984); Abela, G. et al., Circulation, 71(2):403-411 (1985); Prince, N. et al., J. Clin. Invest., 78:295-302 (1986); and Srinivasan, R., Science, 234:559-565 (1986)].
Recent reference has been made to monoclonal antibodies targeting differential antigens in atherosclerotic plaque. These antigens have included oxidized or otherwise modified lipoproteins (Haberland, M. E., et al., Science 241: 215 (1988)) and glycosylated connective tissue proteins (Curtiss, L. K. and Witztum, J. L., J. Clin. Invest, 87: 1436 (1983)). While concentrated within the plaque substance, these antigens have also been found in normal artery and/or other normal tissues. Some antigens and their corresponding monoclonal antibodies have shown early promise in the Watanabe rabbit model but have not held up when applied to human lesions (Shih, I. L., et al., Proc. Natl. Acad, Sci., 87: 1436 (1990)), especially when diffuse markers of extracellular plaque tissue are being sought (Kimura, J., et al., Virchows Arch., 410(2): 159 (1986)).
The subject invention provides an inexpensive, accurate method for determining the presence of atherosclerotic plaque both in vitro and in vivo. In addition, the subject invention provides methods of treating persons having atherosclerotic plaque which include enzyme treatment, and laser treatment. Lastly, the subject invention provides a method of drug delivery to areas of atherosclerotic plaque.